The long-term objective of this project is to measure phosphoproteins in human cancer tissues. As the first step towards this goal, the objective of this R21 grant is to identify optimal conditions for preparation of tissue samples by testing multiple combinations of tissue fixation and protein extraction buffers. Xenograft tissues are frozen or fixed in a non-crosslinking fixative or in formalin. Proteins are extracted with 4 buffers and analyzed for protein phosphorylation using a dot-blot assay. In Aim 1, we measure global phosphorylation on tyrosine and threonine. We also use proQ Diamond to detect all phosphorylated proteins. In Aim 2 we evaluate specific phosphorylation sites in proteins that are of clinical significance as drug targets or predictive biomarkers. We use statistical methods to identify sample preparation protocols that are (1) reproducible, (2) provide a high yield of extracted proteins, and (3) preserve protein phosphorylation during the extraction process. We rank the results and compare the top ranked protocols to a reference sample preparation procedure. We anticipate that by identifying sample preparation protocols for measurement of global and specific protein phosphorylation in cancer tissues, we will improve drug selection and response rates for many patients with solid tumors. [unreadable] [unreadable] Public Health Relevance Statement: Phosphoproteins represent effective predictive and prognostic biomarkers in human cancers; however, their expression is unstable and their measurement difficult. The objective of this project is to test novel sample preparation protocols that may better preserve protein phosphorylation in human cancer tissues for precise and reproducible measurement. [unreadable] [unreadable] [unreadable]